The determination of germs, desired and undesired micro-organisms via microscopy occurred after cultivation on suitable media and by conventional microscopically counting of germs. The use of a microscopy permits to distinguish between micro-organisms and turbidity (e.g. tanning agent), a morphological identification of the micro-organism and a rough estimation of living and dead micro-organism.
This method is known since long and very well described in literature, but bears some disadvantages, such as lack of precision, high degree of manually performed procedures which are difficult to be automated. Furthermore limit is up to 10000 germs/ml.
An alternative microbiological method of the specific determination of germs is based on the growth of those germs on specific media and subsequent determination of the micro-organisms via microscopy. Methods of that kind are commercially available. The application of the specific method however, has substantially the same disadvantages as the above described method for the total count of viable cells. A further disadvantage is the long incubation time of at least 48 hours.
A further method to determine germs is based via biochemical reactions after cultivation on suitable media and total viable count of desired and undesired micro-organism.
Biochemical reactions (as e.g. the API test of Biomerieux) allows distinguishing between different micro-organism. This well known method bears the disadvantage that a high degree of manually performed procedures are necessary, which are difficult to be automated. Furthermore a clear identification of micro-organism is not possible in a mixture of different micro-organisms and cannot be used as a quantitative assay.
Further detection of some micro-organisms may occur by GC/MS or LC/MS by using some secondary metabolites that are synthesized by particular micro-organism. Such metabolites can be detected using a chromatographic separation and a subsequent determination using for example mass spectrometry or spectrophotometrical detection.
One important example in the wine producing industry is for example the repeating detection of the secondary metabolites 4-ethylphenol and 4-ethyl-guajacol which serves as an indication for the presence of Dekkera bruxellensis. As already mentioned above the detection using GC/MS or LC/MS is a rather expensive method. The detection using secondary metabolites cannot be allocated to a unique micro-organism. Furthermore the production of secondary metabolites is strongly dependent on environmental conditions and is therefore not really suitable for quantitative determination.
The newly upcoming PCR Technology (for qualitative use) itself is based on the presence of DNA. The DNA either already exists in single strands or the DNA coil is split into single strands. Two oligonucletide-primers are added in excess. They dock onto a specific part of the DNA not too distant apart from each other and in presence of an initializer for polymerization of nucleotides (polymerase) the specific segment defined/between the primers is replicated. When starting from a single DNA coil, i.e. of two strings, after polymerisation two new coils, i.e. four strings of identical composition are formed. Thereafter those can be used to be replicated in a other cycle. If these steps are reproduced, they lead to an exponential increase of the presence of this specific segment of DNA.
The quantitative PCR method is an improved methodology based on creating an oligonucleotide-probe that fits between the two primers and sits on the segment top be replicated. This methodology is based on the TaqMan®-Technology, which is based on the 5′-nuclease 'PCR assay, published in 1991 by Holland et al., exploiting the 5′-nuclease activity of the TaqPolymerase and the application of fluorescence marked and sequences specific probes.
Those probes are labelled at the 5′-terminal with a fluorescing agent (reporter) and at the 3′-terminal with a fluorescence quencher (quencher).
As the space between them is restricted, the fluorescence emitted by the reporter is quenched by the quencher (or dark quencher) so no fluorescence can be observed. During the polymerase induced chain reaction both the reporter and the quencher or dark quencher, respectively, are released from their positions and are no longer staying close together but are in solution. Under these circumstances, a resulting fluorescence of the reporter can be recorded. The more reporter molecules are released, the higher the intensity of the fluorescence signal results. The quantity of the fluorescence signal is proportional to the amount of sequences replicated. By an analysis of the kinetics, i.e. the number of cycles l needed to obtain a certain signal the initial number of copies of that sequence can be calculated.
This method is extremely sensitive as it replicates the sequences present and hence intensifies the signal to be recorded with each cycle. There are many molecules that show molecules fluorescence under different conditions, which allow to design internal standards that control the success of each replication. Furthermore it is possible to test for the presence of different sequences in parallel using different wavelength to detect the resulting fluorescence of each one.
The optimisation of primer and probe pairs and the variable reaction conditions of the PCR method is essential. The PCR-technology is executed corresponding to the methods described in U.S. Pat. No. 4,683,195, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,800,159.
U.S. Pat. No. 4,683,195 is directed to a process for amplifying and detecting any target nucleic acid contained in a nucleic acid or mixture thereof. The process comprises treating separate complementary strands of the nucleic acid with a molar excess of two oligonucleotide primers, extending the primers to form complementary primer extension products which act as templates for synthesizing the desired nucleic acid sequence, and detecting the sequence so amplified. The steps of the reaction may be carried out stepwise or simultaneously and can be repeated as often as desired.
U.S. Pat. No. 4,683,202 is directed to a process for amplifying any desired specific nucleic acid sequence contained in nucleic acid or mixture thereof.
The process comprises treating separate complementary strands of the nucleic acid with a molar excess of two oligonucleotide primers, and extending the primers to form complementary primer extension products which act as templates for synthesizing the desired nucleic acid sequence. The steps of the reaction may be carried out stepwise or simultaneously and can be repeated as often as desired.
U.S. Pat. No. 4,800,159 claims a process for amplifying and detecting any target nucleic acid sequence contained in a nucleic acid or mixture thereof. The process comprises treating separate complementary strands of the nucleic acid with a molar excess of two oligonucleotide primers in the same manner as described above. In addition, a specific nucleic acid sequence may be cloned into a vector by using primers to amplify the sequence, which contain restriction sites on their non-complementary ends, and a nucleic acid fragment may be prepared from an existing shorter fragment using the amplification process.
U.S. Pat. No. 5,210,015 referring to the three above mentioned US patents is directed to a process of detecting a target nucleic acid using labelled oligonucleotides. This process uses the 5′ to 3′ nuclease activity of a nucleic acid polymerase to cleave annealed labelled oligonucleotide from hybridized duplexes and release labelled oligonucleotide fragments for detection. This can easily be incorporated into a PCR amplification assay.
G. Zapparoli et al. reported in Letters in Applied Microbiology/1998, 27, 243-245 on rapid identification and detection of Oenococcus oeni in wine achieved by specific PCR. Two primers flanking a 1025 bp region of the O. oeni gene encoding the malolactic enzyme were designed. The expected DNA amplificate was obtained only when purified DNA O. oeni was used.
The rapidity and reliability of the PCR procedure established suggests that the method may be profitably applied in winery laboratories for quality control. It has been suggested that the use of above mentioned PCR amplification procedure might also prove useful for the rapid and reliable identification/detection of O. oeni in quality control winery laboratories.
G. Bleve et al. published in Applied and Environmental Microbiology, July, 2003, p. 4116-4122 that reverse transciptase PCR(RT-PCR) and real-time RT-PCR assays have been used to detect and quantify active mRNA from yeast and moulds. Universal primers were designed based on the available fungal actin sequences, and by RT-PCR they amplified a specific 353 bp fragment from species involved in food spoilage, e.g. rapid detection and quantification of viable yeasts and moulds contaminating yoghurts and pasteurised food products were developed, whereas rapid detection and quantification bacteria as micro-organism using actin mRNA have not been used and described yet.
Maria I. Castellanus et al. reported in Current Microbiology Vol. 33 (1996), pp. 100-103 that three lactic acid bacteria previously selected as probiotic for pig feeding, were identified by sequencing the variable V1 region of the 16 S rDNA after PCR amplification primed in the flanking constant region. A VR region showing strong nucleotide differences between the three probiotic and the reference strains was delimited. Oligonucleotides specific for each strain were designed, because this method is not really suitable for differentiation between living and dead microorganism and therefore not suitable for quantification.
Trevor G. Pfister and David A. Mills published in Applied and Environmental Microbiology, December 2003, p. 7430-7434, a Real-time PCR assay for detection and enumeration of spoilage yeast Dekkera bruxellensis in wine without using and describing actin mRNA as target gene, because it is well known that mRNA of ribosomal genes do have a long half-life period.
Casey, Garrett D. et al discloses in International Journal of Food Microbiology 91, 15 Mar. 2004, 327-335, a Real-Time PCR method for detecting and identifying spoilage yeast, such as Saccharomyces cerevisiae in fruit-juice based on the 5.85 rDNA and the adjacent ITS 2 region of these yeasts without using and describing mRNA as target gene. Furthermore the described method is not really suitable to differ between living and dead microorganism, because as already said above it is commonly known that mRNA of ribosamal genes do have a long half-life period.
U.S. Pat. No. 6,248,519, Morenzoni Richard A. et al discloses a PCR method for detecting and identifying a fermentation-related microorganism, such as Saccharomyces cerevisae, in a beverage sample comprising the steps of obtaining DNA from the sample and detecting S. bayanus or S. cerevisae by PCR with primers specific for the S. bayanus or S. cerevisae ITS sequences. This method is also not really suitable for differentiation between living and dead microorganism and therefore not suitable for quantification, because it is known from literature that mRNA ribosomal genes have on the average a long half-life period.
In Database EMBL of Jul. 13, 1992 (1992-07-13) XP002304942 as well as in Database EMBL of Jul. 31, 1991 (1991-07-31) XP002304943 the sequences of Saccharomyces bayanus and Saccharomyces cerevisae have been described, but not the use thereof.
The publication of D. Kosse et al “Identification of yoghurt-spoiling yeast with 18S rRNAY.targeted oligonucleotide porbes” in Systematic and Applied Microbiology, Vol, 20, 1997, pages 468-480 is mainly to be seen as plain state of the art, not being relevant to the invention.